It is well established that inflammation plays an important role in pathogenesis of disease. Inflammation has been shown to be involved in the mechanisms driving neurodegenerative disease, cardiovascular disease, airway disease, inflammatory bowel disease (IBD), diabetes and even cancer. One of the major factors promoting cellular injury during inflammation is oxidative stress. Oxidative stress is induced in cells by over production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and/or an increase in heme peroxidase activity. During inflammation, cells produce highly reactive free radicals (e.g., superoxide anion, hydroxyl radical, peroxide radical, and nitrogen dioxide radical) and strong oxidants (e.g., peroxynitrite and hydrogen peroxide). These free radicals and oxidants have been shown to oxidatively modify proteins, nucleic acids and lipids to the point of causing cell injury and death.
Growing evidence supports the idea that over-expression or increased activity of mammalian heme peroxidases are involved in the pathogenesis and progression of a variety of diseases. For example, increased neutrophil-derived myeloperoxidase (MPO) activity has been found in atherosclerosis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, IBD, kidney disease and rheumatoid arthritis. Another immune cell derived peroxidase, eosinophil peroxidase (EPO), also causes severe respiratory damage in asthma. The relationship between peroxidase and cardiovascular disease is so strong that a measured increase in heme peroxidase (MPO) has been used as a biomarker for the diagnosis and prognosis of cardiovascular disease. In spite of the recent growth in the evidence that heme peroxidases play important roles in the pathogenesis of vascular disease, effective therapies targeting aberrant heme peroxidase activity remain lacking.
Mammalian heme peroxidases, including MPO, are activated by hydrogen peroxide (H2O2). Activated peroxidases catalyze oxidation reactions with a variety of compounds through either a one-electron oxidation cycle or two-electron oxidation cycle. The mechanisms mediating peroxidase activation are as follows. In the inactive native state, the Fe ion in the active site of mammalian heme peroxidases is in the Fe3+ state. This ferrous ion reacts with H2O2 to form compound I, an oxy-ferryl-cation radical (PFe4+═O). Compound I reacts with halides (Cl−1 and Br−1) or pseudohalides (SCN−1) via direct, two-electron reduction to form hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocynite (HOSCN), respectively. As these potent oxidants leave the active site, the heme peroxidases are reduced back to Fe3+ and the cycle starts over again with the arrival of a second H2O2.
Compound I can also react with organic and inorganic substrates such as aromatic amino acids, derivatives of indole and a variety of other species (i.e., nitrite, ascorbate and urate) via two, sequential one-electron reductions. In this reaction sequence, nitrite or tyrosine reduces Compound I to form compound II (Fe4+═O), which yields a nitrogen dioxide radical or a tyrosyl radical, respectively. Compound II can be further reduced by one electron back to the Fe3+ state by a second nitrite or tyrosine.
It is important to note that heme peroxidases can generate both oxidants and free radicals through direct oxidation of biological molecules. This makes them one of the most potent sources of oxidative stress. It has been shown that heme peroxidase-derived oxidants and free radicals oxidatively modify proteins to chlorotyrosine, bromotyrosine, nitrotyrosine, dityrosine, thiol oxidation products and haloamine, DNA molecules to 5-chlorouracil, and lipids to halohydrins, lysophospholipids, α-halo-fatty aldehydes, and other lipid peroxidation products.
As peroxidase-generated oxidants and radicals induce cell injury and death, inhibition of such chronic increases in aberrant peroxidase activity should, in turn, decrease chronic inflammation. Several research programs have worked on developing inhibitors for heme peroxidases over the last several decades. For the most part this research has focused on three lines of investigation. The first line of research focuses on hydrazine (RNHNH2) and hydrazide (RCONHNH2) derivatives that irreversibly inhibit heme peroxidase activity. However, these compounds are considered “suicide substrates” of peroxidase and inactivate the enzyme by destroying the heme center. The second line of research focuses on hydroxamic acids [RCNOHOH or RC(O)NHOH] and indole type compounds that reversibly inhibit peroxidase activity. Hydroxamic acids reduce Compound I and II. They also inhibit H2O2 binding to the heme peroxidase to inhibit formation of Compound I. Some of the indole derivatives (such as tryptophan, tryptamine and melatonin) also rapidly reduce compound I and inhibit the two-electron oxidation cycle of peroxidase. The final line of investigation focuses on another class of compounds that inhibit peroxidase via scavenging heme peroxidase oxidation products, for example, vitamin E and polyphenols scavenge nitrogen dioxide radicals.
Although MPO plays an important role in fighting infection, it is also believed to play a causal role in the development of atherosclerosis. Several clinical studies show that plasma MPO concentrations directly correlate with increased risk of arteriosclerosis, acute myocardial infarction and even heart failure. Immunofluorescent studies show that sickle cell disease (SCD) increases MPO deposition in the subendothelial spaces in the lungs of patients who have died from complications of SCD. The fact that MPO was observed to co-localize with 3-nitrotyrosine in these studies means that when MPO is released and trapped in the vessel wall, it remains fully capable of generating potent oxidants (i.e., .NO2) to nitrate protein tyrosines.
Unfortunately, the progress made in understanding the mechanisms by which MPO impairs vascular function, has not translated into the development of effective therapies. Even though aggressive efforts have been taken, the agents or drugs that have been developed to date fall short for several reasons. Several strategies have been employed. For example, azide, hydrazides and hydroxamic acids have been used to irreversibly inhibit MPO by modifying the heme site. Indole derivatives have been used because they effectively compete with Cl−, SCN− to prevent Compound I from generating HOCl and HOSCN. Phenolic compounds have been used because they effectively scavenge compound I and II.
However, all of these agents have side effects that limit their use to in vitro or cell culture studies. None have been used successfully to reduce oxidative stress in vivo. It is unclear if the intrinsic toxic nature of the compounds (such as hydrazine or hydrazide) or the toxicity of products generated after oxidation by peroxidase makes the compounds essentially worthless as therapeutic agents. In the instances where the agents have been used in animal models, they were observed to be either directly toxic or were converted into toxic compounds. For instance, heme poisons have been shown to inhibit mitochondrial respiration, which is not be a good thing. Even though indole derivatives are effective for scavenging Compound I, MPO oxidizes them to an indole radical that is both toxic and capable of increasing oxidative stress. Finally, even though phenolic agents effectively scavenge Compound I, MPO oxidizes them to phenoxyl radicals that are highly toxic and capable of increasing oxidative stress via oxidative modification of proteins and lipids. Such outcomes underscore the importance of developing specific MPO inhibitors that can be used not only for treating vascular disease but also investigating mechanisms by which MPO increases vascular disease.
Accordingly, there is a long felt but unsolved need in this field to obtain improved agents that effectively prevent or reduce peroxidase-dependent oxidative stress in the in vivo setting.